JP2017520582A - Mesenchymal stromal cells for the treatment of rheumatoid arthritis - Google Patents

Abstract

The present invention provides a method of treating rheumatoid arthritis comprising the use of mesenchymal stromal cells. [Selection figure] None

Description

  The present invention relates to a method for treating rheumatoid arthritis, including the use of mesenchymal stromal cells.

  Rheumatoid arthritis (RA) is an autoimmune disease caused by loss of immunological self tolerance, leading to chronic inflammation of the joint and subsequent cartilage destruction and bone erosion. A very important process underlying the onset of the disease is the induction of autoimmunity against collagen-rich joint components and subsequent events associated with the immune and inflammatory responses that are subsequently deployed. The treatment of rheumatoid arthritis is a major challenge for the health care system in developed countries and increasingly for developing countries. The incidence of rheumatoid arthritis is between 0.5% and 1% of the general population worldwide, with little variation in morbidity by country. Rheumatoid arthritis has more than 21 million patients worldwide (UN World Population Database, 2004) and has a significant impact on the quality of life of patients, resulting in significant economic and social costs. The economic burden associated with the treatment of RA is enormous for the health care economy, and when combined with the annual economic cost of the disease, it is estimated that it will total € 45.5 billion across Europe.

  Traditionally, RA has been treated with non-steroidal anti-inflammatory drugs, glucocorticoids and non-biological DMARDs. Current pharmacological management of rheumatoid arthritis generally involves early intervention using synthetic disease modifying anti-rheumatic drugs (DMARDs) alone or in combination. Where inflammation cannot be adequately suppressed by these means, biological DMARDs that typically target the inflammatory cytokine TNF are utilized. Over the last decade, the introduction of biologics has improved treatment options for patients with rheumatoid arthritis. Biologics, initially intended to neutralize TNF-α, have evolved significantly over the last few years, both in terms of the number of new molecules and the targets they are targeting. Lack of efficacy, non-tolerability or recurrent secondary infections in some patients are factors that require the development of new therapies. Nevertheless, there remains an unmet clinical need for rheumatoid arthritis that approximately 20-40% of rheumatoid arthritis patients do not respond appropriately to anti-TNF.

  Mesenchymal stromal cell therapy has been proposed as an approach for the treatment of inflammatory and autoimmune diseases such as rheumatoid arthritis. Mesenchymal stromal cells (MSCs) are non-hematopoietic stromal cells that can differentiate into mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and fat. MSCs can be easily isolated from tissues such as bone marrow or adipose tissue and rapidly expanded in culture. MSCs can modulate immune responses and exhibit anti-proliferative and anti-inflammatory capabilities, which is the basis for their use as therapeutic candidates for the treatment of immune-mediated inflammatory conditions. However, there is a need to establish effective clinical dosages and dosage regimens for cell therapy in the field of rheumatoid arthritis.

  It has been found that administration of mesenchymal stromal cells (MSC), particularly human adipose tissue-derived stromal cells (hASC), can be useful in the treatment of rheumatoid arthritis.

  Accordingly, the present invention provides a composition comprising mesenchymal stromal cells (MSCs) for use in the treatment of rheumatoid arthritis in a subject, eg, for use in a method of treating rheumatoid arthritis in a subject. To do.

  The present invention also provides the use of mesenchymal stromal cells (MSCs) in the manufacture of a medicament for treating rheumatoid arthritis in a subject.

  The present invention also provides a method of treating rheumatoid arthritis in a subject in need thereof, comprising administering to the subject a composition containing mesenchymal stromal cells.

  In one aspect, the invention provides a human subject with moderate rheumatoid arthritis having a CDAI score> 10 to ≦ 22 and optionally having a DAS28 score> 3.2 to ≦ 5.1 and / or a RAPID3 score> 6 to ≦ 12. Compositions containing mesenchymal stromal cells (MSCs) for use in the treatment of rheumatoid arthritis are provided.

The arthritis score (A) and incidence (B) 21-54 days after collagen injection are shown. “CIA” = no treatment; “CIA + ASC d15, d18, d21” = intravenous injection of ASC at 15, 18 and 21 days after collagen injection. Shown are arthritis scores on days 1-59 (Day 1 = first ASC administration). ASC was administered intravenously only on days 1, 3 and 5 or subsequently on days 8, 15, 22 and 29. The effect of three intravenous doses on days 1, 3 and 5 (Day 1 = initial ASC administration) of ASC (8,000, 40,000, 200,000 or 1,000,000 ASCs per dose) is shown. The effect of three intravenous doses on days 1, 8 and 15 (Day 1 = initial ASC administration) of ASC (8,000, 40,000, 200,000 or 1,000,000 ASCs per dose) is shown. (A) IL6 on day 22 in healthy mice, untreated arthritic mice (“CIA”), and arthritic mice treated with ASC by intravenous administration (“CIA + ASC IV”); (B) IL17; C) IL23; and (D) CD3 + CD25bright (regulatory T cells) levels. Day 50 bone mineral density (A) and trabecular mineral density (B) in untreated arthritic mice (“CIA”) and arthritic mice treated with ASC by intravenous administration (“CIA + ASC”) ). Figure 5 shows bone mineral density (A) and trabecular mineral density (B) at day 50 in untreated arthritic mice (left bar) and arthritic mice treated with ASC by intralymphatic administration (right bar). The effect of intralymphatic administration in early arthritis is shown. Arthritis score (A and B) and foot volume (C and D). Regulatory T cells (CD4 + CD25 + FoxP3 + cells) in spleen (A) and lymph nodes (B) of healthy mice, untreated arthritic mice (“CIA”), and arthritic mice after intravascular administration of ASC (“CIA + ASC IL”) ) Level. IL10-expressing CD4 cells (TR1) in spleens (A) and lymph nodes (B) of healthy mice, untreated arthritic mice (“CIA”), and arthritic mice after intravascular administration of ASC (“CIA + ASC IL”) Cell) level. ASC-treated human patients starting at DAS28 <4.5 (lower limit) or> 5.5 (upper limit) at baseline (A) and normalized to baseline (B), recorded over several visits (V4-V9) Shows DAS28 response in subpopulation. ASC in baseline (A) and baseline normalized (B) starting from CDAI <22 (lower limit) or> 22 and <40 (upper limit) recorded over several visits (V4-V9) CDAI levels in treated human patient subpopulations are shown. ASC treated patients (ITT population) that achieved ACR20 (A), ACR50 (B), and ACR70 (C) throughout the study. Good EULAR response throughout the study (A); low disease activity (DAS28-ESR <3.2) (B); clinical remission (DAS28-ESR <2.6) (C); and DAS28-ESR change from baseline throughout the study (D) ASC-treated patients (ITT population).

Definitions As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

  The articles “a” and “an” mean one, or more than one (ie, at least one) grammatical object. For example, “an element” means one element or more than one element.

  The term “about”, when used in connection with a numerical value, relates to the numerical value ± 10%.

  “Adipose tissue” means any fat tissue. The adipose tissue can be, for example, brown or white adipose tissue from the subcutaneous, omental / visceral, mammary, gonadal, or other adipose tissue site. Preferably, the adipose tissue is subcutaneous white adipose tissue. Adipose tissue can include primary cell cultures or immortalized cell lines. The adipose tissue can be from any organism that has fat tissue. In some embodiments, the adipose tissue is from a mammal, and in further embodiments, the adipose tissue is from a human. A convenient source of adipose tissue is liposuction surgery. However, it will be understood that neither the source of adipose tissue nor the method of isolation of adipose tissue is critical to the present invention. If the cells described herein are desired for autotransplantation into a subject, adipose tissue will be isolated from the subject.

  “Adipose tissue-derived stromal cells (ASC)” refers to MSCs derived from adipose tissue, generally human adipose tissue (hASC).

  “Resistance” means that there is no significant clinical benefit when used in the treatment of a disease, for example, no significant improvement or alleviation of symptoms, relapse or toxicity following a disease, etc. It should be understood as meaning.

  The term “biopharmaceutical” is a protein or nucleic acid base for therapeutic use, typically manufactured by means other than direct extraction from a natural (non-engineered) biological source. It should be understood as meaning a medicinal substance.

  The term “cells / kg” as used herein should be understood to mean the number of cells (eg, MSCs) administered per kg patient body weight.

  The term “constitutive” is understood to mean the expression of a gene without any special induction.

  The term “culture” refers to the growth of cells, organisms, multicellular material, or tissues in a medium. The term “culturing” refers to any method of achieving such growth and can include multiple stages. The term “further culture” refers to culturing a cell, organism, multicellular material, or tissue to a specific stage of growth, and then using another culture method to propagate the cell, organism, multicellular material, or tissue. Incubation until another stage. “Cell culture” refers to the growth of cells in vitro. In such a culture, the cells proliferate but do not structure themselves to the tissue. “Tissue culture” refers to the maintenance or growth of a graft in vitro in a primary or adult organ, such as one that maintains its structure and function. “Monolayer culture” refers to a culture in which cells grow in a suitable medium while primarily adhering to each other and to the substrate. Furthermore, “suspension culture” refers to a culture in which cells grow while suspended in a suitable medium. Similarly, “continuous flow culture” refers to culturing cells or grafts in a continuous flow of fresh medium to maintain cell growth, eg, survival. The term “conditional medium” is obtained after a certain period of time in contact with cultured cells where the medium has been modified to contain certain paracrine and / or autocrine factors produced by the cells and secreted during culture. For example, a supernatant containing no cultured cells / tissues. “Confluent culture” is a cell culture in which all cells are in contact and thus the entire surface of the culture vessel is covered, meaning that the cells have reached their maximum density, but confluence does not necessarily stop dividing. Does not mean that the group will not grow.

  The term “culture medium” or “medium” is art-recognized and refers to any substance or preparation generally used for culturing living cells. The term “medium” as used in connection with cell culture includes components of the environment surrounding the cells. The medium may be solid, liquid, gaseous or a mixture of phases and substances. Media include liquid growth media as well as liquid media that do not maintain cell growth. The medium also includes gelled medium such as agar, agarose, gelatin and collagen matrix. Examples of gaseous media include the gas phase to which cells growing on Petri dishes or other solid or semi-solid supports are exposed. The term “medium” also refers to a substance that is intended to be used in cell culture, even if it has not yet come into contact with the cells. In other words, a nutrient rich liquid prepared for bacterial culture is a medium. Similarly, a powder mixture that becomes suitable for cell culture when mixed with water or other liquids may be referred to as a “powdered medium”. “Defined medium” refers to a medium composed of chemically defined (usually purified) components. The “defined medium” does not include biological extracts that are not well characterized, such as yeast extract and beef broth. A “rich medium” includes media that are designed to support the growth of most or all viable forms of a particular species. Enriched media often contain complex biological extracts. A “medium suitable for high density culture growth” allows cell cultures to reach an OD600 of 3 or more when other conditions (eg temperature and oxygen transfer rate) allow such growth. Any medium. The term “basic medium” refers to a medium that promotes the growth of many types of microorganisms that do not require any specific nutritional supplementation. Most basal media typically contain four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. Basal media generally serve as the basis for more complex media to which supplements such as serum, buffers, growth factors, lipids, etc. are added. Examples of basal media include, but are not limited to, Eagle basal medium, minimal essential medium, Dulbecco's modified Eagle medium, medium 199, nutrient mixture Ham's F-10 and Ham's F-12, McCoy's 5A, Dulbecco MEM / FI 2 , RPMI 1640, and Iskov modified Dulbecco's medium (IMDM).

  The terms “comprise” and “comprising” are used in an inclusive, open sense, meaning that additional elements may be included.

  The term “expanded” as used herein in relation to cells should be understood as having the usual meaning in the art, ie cells grown in vitro. MSCs can be expanded to provide a population of cells that retains at least one biological function of MSCs, typically the ability to adhere to plastic surfaces under standard culture conditions. An expanded population of cells can retain the ability to differentiate into one or more cell types.

  The term “including” is used herein to mean “including but not limited to”. “Including” and “including but not limited to (including but not limited to)” are used interchangeably.

  “Marker” refers to a biological molecule that can be used to detect its presence, concentration, activity, or phosphorylation status to identify the phenotype of a cell.

  “Mesenchymal stromal cells” (also referred to herein as “MSCs”) are pluripotent stromal cells, ie, cells that can give rise to a plurality of different types of cells.

  A “patient”, “subject” or “host” to be treated by the method can mean either a human or non-human animal.

  The term “pharmaceutical composition” refers to a composition intended for use in therapy. The composition of the present invention is a pharmaceutical composition intended for use in the treatment of rheumatoid arthritis. The composition of the present invention may contain non-cellular components in addition to MSC. Examples of such non-cellular components include, but are not limited to, cell culture media that can include one or more of proteins, amino acids, nucleic acids, nucleotides, coenzymes, antioxidants, and metals.

  The term “pharmaceutically acceptable” is used herein to account for excessive toxicity, irritation, allergic response, or other problems, within reasonable medical judgment and in proportion to a reasonable benefit / risk ratio. Used to refer to compounds, substances, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without complications.

  The term “pharmaceutically acceptable carrier” as used herein refers to a liquid or solid involved in carrying or transporting a compound of interest from one organ or body part to another organ or body part. It means a pharmaceutically acceptable substance, composition or vehicle, such as a filler, diluent, excipient, or solvent-encapsulated substance. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

  The term “phenotype” refers to the observable properties of a cell, such as size, morphology, protein expression.

  “Proliferation” refers to an increase in the number of cells. “Proliferating” and “proliferation” refer to cells undergoing mitosis.

  “Resistant”, when used in the treatment of a disease, means having no significant clinical benefit, eg, no significant improvement or alleviation of symptoms, or subsequent recurrence of the disease Should be understood.

  As used herein, the term “solution” includes a pharmaceutically acceptable carrier or diluent in which the MSC used in the present invention can survive.

  The term “substantially pure” as used with respect to an MSC population refers to a population of cells in which at least about 75%, at least about 85%, at least about 90%, or at least about 95% of the number of cells are MSCs. In other words, the term “substantially pure” as used with respect to an MSC population refers to a population of cells that contain less than about 20%, less than about 10%, or less than about 5% of the cells whose lineage is restricted. .

  As used herein, “support” refers to any device or substance that can function as a foundation or matrix for the growth of adipose tissue-derived stromal cells.

  “Therapeutic agent” or “therapeutic agent” refers to an agent that can have a desired biological effect on a host. Chemotherapeutic agents and genotoxic agents are examples of therapeutic agents that are generally known to be of chemical origin, unlike biologics, or that produce a therapeutic effect by a specific mechanism of action. Examples of therapeutic agents of biological origin include growth factors, hormones, and cytokines. Various therapeutic agents are known in the art and can be identified by their effects. Certain therapeutic agents can modulate cell proliferation and differentiation. Examples include chemotherapeutic nucleotides, drugs, hormones, non-specific (non-antibody) proteins, oligonucleotides (eg, antisense oligonucleotides that bind to target nucleic acid sequences (such as mRNA sequences)), peptides, and peptidomimetics. It is done.

  As used herein, an “initial diagnosis” is preferably the first by a qualified physician in rheumatoid arthritis (eg, rheumatologist, general practitioner, MD, nurse practitioner), based on clinically accepted criteria. Should be understood to mean the diagnosis of

  “Disease period” should be taken to mean the time since the onset of the disease.

The present invention relates to a novel use of mesenchymal stromal cells (hereinafter also referred to as “MSC”) in the treatment of inflammatory joint diseases, and a composition of mesenchymal stromal cells suitable for such use and preparation thereof. Provide a method. In one embodiment, the inflammatory joint disease is rheumatoid arthritis. In one embodiment, the inflammatory joint disease is progressive or polycyclic rheumatoid arthritis. In one embodiment, the inflammatory joint disease is moderate or severe rheumatoid arthritis.

  Diagnosis and severity classification is preferably clinically accepted criteria such as those established by the American College of Rheumatology (ACR) or the European League Against Rheumatism (EULAR), eg Disease Activity Score 28-Disease Activity Score 28-joint count (DAS28), Clinical Disease Activity Index (CDAI), and Routine Assessment of Patient Index Data 3 , Using RAPID3). In one embodiment, a DAS28 score> 3.2 to ≦ 5.1 is considered moderate and a DAS28 score> 5.1 is considered severe. In another embodiment, a CDAI score> 10 to ≦ 22 is considered moderate and a CDAI score> 22 is considered severe. In yet another embodiment, a RAPID3 score> 6 to ≦ 12 is considered moderate and a RAPID3 score> 12 is considered severe.

  Thus, the present invention provides an MSC for use in the treatment of moderate rheumatoid arthritis in a patient, wherein the disease has a DAS28 score> 3.2 to ≦ 5.1, a CDAI score> 10 to ≦ 22, and And / or RAPID3 score> 6 to ≤12.

  Such patients typically have “early rheumatoid arthritis”. Historically, early rheumatoid arthritis was considered a disease of less than 5 years, but by the early 1990s, this was lowered to less than 24 months, with the first 12 months being more important became. Currently, many rheumatologists want to see early RA patients on the first occasion. The proportion of rheumatologists who want to see patients within 6 weeks of the onset of symptoms has doubled from 9% in 1997 to 17% in 2003, but all patients are being examined so early (DL Scott, Early rheumatoid arthritis, British Medical Bulletin 2007; 81 and 82: 97-114).

  Accordingly, the present invention also provides an MSC for use in the treatment of moderate rheumatoid arthritis in a patient, wherein the disease has a CDAI score> 10 to ≦ 22, optionally a DAS28 score> 3.2 to ≦ 5.1. And / or RAPID3 score> 6 to ≦ 12 and rheumatoid arthritis is early rheumatoid arthritis, for example within about 6 months from the onset of the first diagnosis or disease symptoms, or about 12 months from the onset of the first diagnosis or disease symptoms Is within. The examples show that particularly good therapeutic effects are achieved in this subpopulation.

  The invention also provides an MSC for use in the treatment of severe rheumatoid arthritis in a patient, wherein the disease has a DAS28 score> 5.1, a CDAI score> 22 and / or a RAPID3 score> 12.

  In a preferred embodiment of the invention, MSC is within about 6 months from the first diagnosis, within about 12 months from the first diagnosis, within about 18 months from the first diagnosis, within about 2 years from the first diagnosis, from the first diagnosis Used in the treatment of rheumatoid arthritis in patients within about 3 years, within about 4 years from the first diagnosis, and within about 5 years from the first diagnosis.

  In a preferred embodiment of the invention, the MSC has a disease duration of about 6 months, a disease duration of about 12 months, a disease duration of about 18 months, a disease duration of about 2 years, a disease duration of about 3 years, Used in the treatment of rheumatoid arthritis in patients with a disease duration of about 4 years or less, and a disease duration of about 5 years or less.

  Typically, MSCs are used in the treatment of rheumatoid arthritis in patients in the acute phase of the disease.

  The term “acute phase” in the context of rheumatoid arthritis should be understood to mean a patient experiencing significant inflammation of one or more joints, rather than only mild or moderate inflammation. The acute phase is also referred to in the art as “flares”.

  Methods for diagnosing acute rheumatoid arthritis are known in the art and include analysis of erythrocyte sedimentation rate (ESR) and / or serum C-reactive protein (CRP) levels, eg, ESR above 50 is considered acute. It is.

  MSC has been found to confer an immunomodulatory effect, especially at sites of inflammation. During acute flares of rheumatoid arthritis characterized by elevated inflammatory markers (ie during the acute phase), externally delivered MSCs are mediated by modulation of the inflammatory immune response and the resulting suppression of the inflammatory cytokine environment It is a therapeutic agent. MSC is therefore particularly effective in the treatment of rheumatoid arthritis in the acute phase before the irreversible process is established. Thus, the present invention provides MSCs for use in the treatment of rheumatoid arthritis in patients in the acute phase of the disease.

  In a preferred embodiment of the invention, MSCs are used in the treatment of rheumatoid arthritis in patients who are resistant to at least one, two or three therapies for the treatment of rheumatoid arthritis. Typically, the patient will receive the above treatment for at least about 6, 12, 18, 24 or 36 weeks and still exhibit symptoms of active disease. In other words, even after at least about 6, 12, 18, 24 or 36 weeks of treatment, the severity of the disease is not reduced.

  The therapy may include one or more of disease modifying anti-rheumatic drugs (DMARD), non-steroidal anti-inflammatory drugs (NSAIDs), biopharmaceuticals and corticosteroids. NSAIDs include, but are not limited to, Cox-2 inhibitors, diclofenac, ibuprofen, naproxen, celecoxib, mefenamic acid, etoricoxib, indomethacin and aspirin. Corticosteroids include, but are not limited to, triamcinolone, cortisone, prednisone, and methylprednisolone. Disease modifying anti-rheumatic drugs (DMARDs) include but are not limited to auranofin, chloroquine, cyclosporine, cyclophosphamide, gold preparation, hydroxychloroquine sulfate, leflunomide, methotrexate, penicillamine, Examples include sodium aurothiomaleate and sulfasalazine.

  In one embodiment, the patient is preferably at least 2 or 3 DMARDs selected from the group consisting of hydroxychloroquine, leflunomide, methotrexate, minocycline, and sulfasalazine (eg, methotrexate and hydroxychloroquine; methotrexate and leflunomide; methotrexate and sulfasalazine). Sulfasalazine and hydroxychloroquine; methotrexate, hydroxychloroquine and sulfasalazine).

  These agents target specific anti-inflammatory molecular pathways. In contrast, MSCs provide a more physiological approach and involve regulating multiple inflammatory pathways and suppressing pathological excessive inflammatory responses. MSCs also provide a mechanism for negative feedback loops that provide a physiological balance between pro- and anti-inflammatory cytokines, but this is not possible with mere delivery of a predetermined amount of anti-inflammatory drug. is there. Thus, the MSCs of the present invention are particularly effective in the treatment of rheumatoid arthritis in patients who are refractory to any anti-inflammatory drug. In one embodiment, it is particularly preferred that the patient is at least resistant to treatment with methotrexate. In one embodiment, the patient is resistant to at least two or three DMARDs, including methotrexate and one or more DMARDs selected from the group consisting of hydroxychloroquine, leflunomide, minocycline, and sulfasalazine. It is.

  In further embodiments, the patient is treated with a combination therapy comprising methotrexate and at least one, two or all of a corticosteroid, a nonsteroidal anti-inflammatory agent, and a DMARD (eg, leflunomide and / or sulfasalazine), eg, methotrexate. And prednisone; or methotrexate, sulfasalazine and prednisone. In one embodiment, it is particularly preferred that the patient is at least resistant to treatment with leflunomide. In a further embodiment, it is preferred that the patient is at least resistant to treatment with both leflunomide and methotrexate.

  In a further embodiment, the patient is preferably resistant to treatment with methotrexate and cyclosporine. These patients are likely to be non-responders to TNF therapy.

  In further embodiments, the patient is treated with a combination therapy comprising methotrexate, leflunomide, and at least one, two or all of corticosteroids, non-steroidal anti-inflammatory drugs, and sulfasalazine, such as methotrexate, leflunomide, DMARD (e.g. Hydroxychloroquine and / or sulfasalazine) and corticosteroids (eg prednisone) are preferably resistant to treatment.

  Typically, the patient has been treated with at least methotrexate for at least about 6, 12, 18, 24, or 36 weeks and has an active disease. In other words, even after at least about 6, 12, 18, 24 or 36 weeks of treatment, the severity of the disease has not been reduced.

  In some cases, the patient may have been administered a biopharmaceutical. Biopharmaceuticals include, but are not limited to, selective costimulatory modulators, TNF-α inhibitors, IL-1 inhibitors, IL-6 inhibitors, and B cell targeted therapeutic agents. Typically, a patient is resistant to at least one biological therapy, such as a TNF-α inhibitor. Thus, in one embodiment of the invention, the patient is at least a TNF-α inhibitor, typically adalimumab (Fumila), certolizumab (Simdia), etanercept (Enbrel), golimumab (Simponi), or infliximab (Remicade) Resistant to one type of biological treatment. Typically, a patient is resistant to at least two biological treatments, optionally at least one of which can be a TNF-α inhibitor. In one embodiment, the patient is at least resistant to methotrexate and a TNF-α inhibitor, wherein the TNF-α inhibitor is particularly preferably selected from the group comprising adalimumab, etanercept and infliximab. .

  In further embodiments, it is particularly preferred that the patient is at least resistant to treatment with leflunomide and a TNF-α inhibitor, wherein the TNF-α inhibitor is selected from the group comprising adalimumab, etanercept and infliximab. In a further embodiment, the patient is at least resistant to treatment with leflunomide, methotrexate and a TNF-α inhibitor, wherein the TNF-α inhibitor is selected from the group comprising adalimumab, etanercept and infliximab. preferable.

  The present invention also provides an MSC for use in the treatment of rheumatoid arthritis in patients in the acute phase of a disease that is refractory to methotrexate. Preferably, the patient is further resistant to treatment with a DMARD, such as cyclosporine.

  The examples show that arthritis can be treated by administering ASC and that the therapeutic effect is particularly good when ASC is initially administered in the early / mid-stage stages of the disease. Surprisingly, useful long-term therapeutic effects are observed even after discontinuing treatment with ASC. Benefit is observed over the entire period of ASC administration, eg 3 or 7 administrations separated by 1 day or 1 week (FIGS. 1-4). Repeated administration of ASC, e.g. 3 doses at 1 day or 1 week intervals, has been shown to be beneficial in human patients, especially those with moderate rheumatoid arthritis, even 6 months after ASC administration (FIG. 12).

  Thus, in one embodiment, the invention provides that MSCs are administered repeatedly, i.e., two or more doses, e.g. at least 3, doses, at least 4, 5, 6, 7, 8, 9, or 10 An MSC is provided for use in the treatment of rheumatoid arthritis in a subject to which a single dose is administered. The interval between doses can be daily, weekly, or monthly. MSCs can be used for periods of less than one week (eg every other day, such as day 1 and day 3, or day 1, day 3 and day 5, or day 1, day 3, day 5 and day 7 Or MSC can be administered for a period of 1 week or longer (eg day 1, day 8 and day 15, where day 1 represents the first dose of MSC), eg at least 4 weeks, at least 1 month, at least 6 weeks, at least 8 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months, or at least 12 months Can be administered for a while. In each of these embodiments, rheumatoid arthritis has a CDAI score> 10 to ≦ 22, optionally a DAS28 score> 3.2 to ≦ 5.1, and / or a RAPID3 score as measured at baseline (ie at the first ASC administration)> It may be moderate rheumatoid arthritis with 6 to ≦ 12. Typically, the MSC is an ASC, such as human eASC, optionally administered intralymphatic, for example by intralymphatic infusion.

  The example also shows that treatment with ASC significantly reduces the levels of pro-inflammatory cytokines IL6, IL17, and IL23 and increases the level of regulatory T cells (CD3 + CD25bright) compared to untreated mice (FIG. 5). Thus, in one embodiment, the invention provides an MSC for use in the treatment of rheumatoid arthritis in a subject, wherein administration of MSC is systemic IL6, IL17 compared to an untreated subject. And / or the level of IL23 is reduced to a statistically significant level (eg, as measured in plasma of peripheral blood; related methods such as ELISA are well known to those skilled in the art). In some embodiments, the systemic level of IL6, IL17 and / or IL23 is 80% or less, such as 75% or less, 70%, compared to typical systemic levels of individual cytokines in an untreated subject. Below, 65% or less, 60% or less, 55% or less, or 50% or less. Alternatively, or in addition, in some embodiments, the level of systemic CD3 + CD25bright cells compared to an untreated subject (eg, as measured in a cellular fraction of peripheral blood; For example, FACS is well known to those skilled in the art) and rises to a statistically significant level. In some embodiments, the level of systemic CD3 + CD25bright cells is 2 fold or more, eg, 2.5 fold or more, compared to the typical systemic level of CD3 + CD25bright cells in an untreated subject.

  Intralymphatic administration has been shown to be useful (Figure 8). Thus, in any of the above embodiments, MSC, typically ASC, can be administered by an intralymphatic route, such as intralymphatic infusion.

Cell of the present invention
MSCs are undifferentiated stromal cells that have the ability to differentiate into other cells, typically derived from connective tissue and are therefore non-hematopoietic cells. The term “connective tissue” refers to tissue derived from mesenchyme and includes a number of tissues characterized in that their cells are contained within the extracellular matrix. Among the different types of connective tissue are adipose tissue and cartilage tissue. In one embodiment, the MSC is derived from the stromal fraction of adipose tissue. In another embodiment, the MSC is derived from chondrocytes, such as hyaline cartilage. In another embodiment, the MSC is obtained from the skin. In yet another embodiment, the MSC is obtained from bone marrow. Alternative sources of MSC include, but are not limited to, periosteum, pulp, spleen, pancreas, ligament, tendon, skeletal muscle, umbilical cord and placenta.

  MSCs can be obtained from any suitable source and from any suitable animal, including humans. Typically, the cells are obtained from mammalian connective tissue after birth. In a preferred embodiment, MSCs are obtained from a source of connective tissue, such as a stromal fraction such as adipose tissue, hyaline cartilage, bone marrow, skin and the like. Also, in certain embodiments, the MSC is from a mammal, such as a rodent, a primate, etc., preferably a human. Typically, MSCs are obtained from the stromal fraction of human adipose tissue. That is, they are adipose tissue-derived stromal cells (ASC).

  MSCs are adhesive to plastics under standard culture conditions.

  MSCs differentiate into or in somatic cells such as mesoderm cells (eg adipocytes, chondrocytes, osteoblasts) and optionally in endoderm and / or ectoderm cell types or lineages or in that direction. It is an undifferentiated pluripotent cell having the ability to Typically, the cells have the ability to differentiate into or in at least two or all cell types selected from the group consisting of adipocytes, chondroblasts and osteoblast lineages.

  In one embodiment, the MSC can be a stem cell, typically an adipose tissue-derived stem cell. Typically, MSCs (i) do not express markers specific for APC, (ii) do not constitutively express IDO, and (iii) do not constitutively significantly express MHC II. Typically, IDO or MHC II expression can be induced by stimulation with IFN-γ.

  Typically, the MSC is one or more of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 (eg, 2 or more, 3 or more, 4 or more) 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 13) can be expressed. For example, the MSC can express one or more of the markers CD29, CD59, CD90 and CD105 (eg, two, three or all), eg, CD59 and / or CD90.

  Typically, MSC is one of the markers Factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133 Does not express more than 2 species (eg 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 15)) . For example, MSCs do not express one or more of the markers CD34, CD45, CD31 and CD14 (eg, two, three or all), eg, CD31 and / or CD34.

Increased MSC
In one embodiment, the MSC is an MSC increased in in vitro culture or a progeny cell increased in in vitro culture (hereinafter both referred to as increased MSC or “eMSC”). Methods for preparing eMSC are known in the art, for example as described in WO2007 / 039150. eMSC retains several characteristic phenotypes of MSC. For example, eMSCs adhere to plastics under standard culture conditions and retain an undifferentiated phenotype.

  eMSCs are undifferentiated pluripotent cells that have the ability to differentiate into somatic cells such as mesoderm cells. While MSCs have the ability to differentiate towards at least one or more specialized cell lines such as but not limited to adipocytes, chondroblasts, osteoblast lineages, etc., eMSC typically Differentiating ability may be reduced or even lost. For example, MSCs can differentiate towards at least osteogenic and adipocyte lineages, while eMSCs derived from them can only differentiate towards adipocyte lineages. This is advantageous for therapeutic applications of cells where cells can be administered to patients, as it can reasonably be predicted that the potential for unexpected and potentially harmful differentiation of eMSCs is low. obtain.

  In one embodiment, the eMSC can be a progeny of stem cells. Typically, eMSCs (i) do not express APC specific markers; (ii) do not constitutively express IDO; (iii) do not constitutively significantly express MHC II. Typically, IDO or MHC II expression can be induced by stimulation with IFN-γ.

  Typically, eMSC is one or more of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 (eg, 2 or more, 3 or more, 4 or more) 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 13) can be expressed. For example, the MSC can express one or more of the markers CD29, CD59, CD90 and CD105 (eg, two, three or all), eg, CD59 and / or CD90.

  Typically, eMSC is one or more of the marker factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133 ( For example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 15)) are not expressed. For example, MSCs do not express one or more of the markers CD34, CD45, CD31 and CD14 (eg, two, three or all), eg, CD31 and / or CD34. Furthermore, MSCs sometimes do not express the marker STRO-1.

In one embodiment of the cell population , the present invention provides populations of MSCs and / or eMSCs for therapeutic uses as described herein, these populations are hereinafter referred to as “cell populations of the invention”. Sometimes called. Typically, the MSC is increased human ASC, typically allogeneic increased human ASC. Typically, the cell population of the present invention is a homogeneous or substantially homogeneous population of MSCs and / or eMSCs. Although the cell populations of the present invention comprise or essentially comprise MSCs and / or eMSCs, the cell populations of the present invention can also comprise other cell types. Accordingly, in one embodiment, the invention provides at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about at least about 25% of the cells. 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cell populations of the invention are provided that are MSCs and / or eMSCs.

  Typically, the cell population of the present invention is a population expanded with cultures of MSCs comprising or essentially comprising eMSCs, although the cell populations of the present invention may comprise other cell types. Accordingly, in one embodiment, the invention provides at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about at least about 25% of the cells. 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cell populations of the present invention are provided. In one embodiment, the eMSC is eASC, such as human eASC. In certain embodiments, the cells are allogeneic to the subject being treated.

  Typically, the cell populations of the present invention are directed towards at least one or more specialized cell lines such as, but not limited to, adipocyte lineage, chondroblast lineage, and osteoblast lineage. May have the ability to differentiate. In one embodiment, the cell population of the present invention is directed towards or towards at least two or all cell types selected from the group consisting of adipocyte, chondroblast, and osteoblast cells. May have the ability to differentiate. However, in other embodiments, this differentiation potential may be reduced or even gone. For example, MSCs can differentiate at least towards osteogenic and adipocyte systems, while eMSC populations derived therefrom can only differentiate towards adipocyte systems. This can be advantageous when cells can be administered to patients because it can reasonably be expected that there will be less chance of differentiation that could be unexpectedly harmful to eMSC in therapeutic applications of cells. .

  Typically, the cell population of the present invention comprises one or more of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 (eg, two or more, three types). As described above, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 13) can be expressed. For example, the MSC can express one or more of the markers CD29, CD59, CD90 and CD105 (eg, two, three or all), eg, CD59 and / or CD90. Accordingly, in one embodiment, the invention provides at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about at least about 25% of the cells. 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% is one or more of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 (eg 2 or more, 3 or more, 4 Provided is a cell population of the present invention that expresses at least 5 species, 5 species, 6 species, 7 species, 8 species, 9 species, or 10 species (for example, up to 13 species). For example, the MSC can express one or more of the markers CD29, CD59, CD90 and CD105 (eg, two, three or all), eg, CD59 and / or CD90. In another embodiment, the present invention provides one or more of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 (eg, two or more, three or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 13) expression levels of at least about 25%, at least about 30%, at least About 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least Cell populations of the invention that are about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% are provided. For example, the cell population of the present invention can express one or more (eg, two, three or all) of the markers CD29, CD59, CD90 and CD105, eg, CD59 and / or CD90 at the above levels.

  Typically, the cell population of the present invention comprises marker factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133. 1 or more (for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 15)) It does not have to be expressed. For example, MSCs do not express one or more of the markers CD34, CD45, CD31 and CD14 (eg, two, three or all), eg, CD31 and / or CD34. Furthermore, the MSC may optionally not express the marker STRO-1.

  Accordingly, in one embodiment, the invention provides at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about at least about 25% of the cells. 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% is one of the markers Factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133 Books that do not express the above (for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 15)) Inventive cell populations are provided. For example, the MSC may be one that does not express one or more of the markers CD34, CD45, CD31 and CD14 (eg, two, three or all), eg, CD31 and / or CD34. In another embodiment, the present invention relates to one of the marker factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133. Expression level of 2 or more species (eg 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (for example, up to 15)) A cell population of the present invention wherein is at least about 35% or less, at least about 30% or less, at least about 25% or less, at least about 20% or less, at least about 25% or less, at least about 5% or less, at least about 1% or less provide. For example, the cell population of the invention can express one or more of the markers CD34, CD45, CD31 and CD14 (eg, two, three or all), eg, CD31 and / or CD34 at the levels described above.

  In some embodiments, the MSC or eMSC is pre-stimulated to increase one or more of its proliferative ability, migration ability, viability, therapeutic effect and immunomodulation. Typically, at least about 40% (eg, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the cell population of the invention. %, At least about 96%, at least about 97%, at least about 98%, or at least about 99%) in advance to increase one or more of its proliferative ability, migration ability, viability, therapeutic effect and immunomodulation Irritate. In some embodiments, the pre-stimulation can be achieved by contacting the MSC with a cytokine. In some embodiments of the invention, the pre-stimulation can be achieved by contacting the MSC with IFN-γ.

In one embodiment, the present invention provides a composition comprising a cell population of the present invention for use in a therapeutic method according to the present invention. The composition is preferably a pharmaceutical composition. The composition of the invention may comprise a substantially pure population of MSC or eMSC, such as a substantially pure population of ASC or eASC, eg human eASC. The MSC, eMSC, ASC or eASC can be a stem cell. The composition of the present invention may also include a culture medium containing cell culture components, such as one or more amino acids, metals and coenzyme factors. The composition can include a small population of other stromal cells. The composition may also include other non-cellular components that can support the growth and survival of MSCs in certain circumstances, such as transplantation, growth in continuous culture, or use as a biomaterial or composition.

The concentration of MSC and / or eMSC in the composition of the present invention is at least about 1 × 10 ⁴ cells / mL, at least about 1 × 10 ⁵ cells / mL, at least about 1 × 10 ⁶ cells / mL, at least about 10 ×. It can be 10 ⁶ cells / mL, or at least about 40 × 10 ⁶ cells / mL. Typically, the concentration is between about 1 × 10 ⁶ cells / mL and 1 × 10 ⁷ cells / mL, such as between about 5 × 10 ⁶ cells / mL and 1 × 10 ⁷ cells / mL.

  The compositions of the invention will generally comprise a pharmaceutically acceptable carrier and / or diluent. Examples of such carriers and diluents are well known in the art and may include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose , Ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; For example, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-removing water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solution; polyesters, polycarbonates and / or polyanhydrides; Non-toxic compatible substance used in the formulation. Typically, the compositions of the present invention contain DMEM as a carrier, which optionally contains serum (eg, HSA), for example up to about 5%, up to about 10%, up to about 15%, up to about 20%. Can be added at a concentration of up to about 25%, up to about 30%.

  The compositions of the present invention may be fluid to the extent that is aseptic and / or easy to pass through a syringe. Furthermore, the composition may be stable under the conditions of manufacture and storage and / or protected from contamination of microorganisms such as bacteria and fungi, for example, through the use of parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.

Preparation Methods Methods for isolating and culturing MSCs to provide eMSCs and cell populations of the invention and compositions comprising the cell populations of the invention are known in the art. Typically, a method for preparing a composition comprising a cell population comprises the following steps:
(i) Isolation of MSCs from tissue and selection by adhesion to a suitable surface, eg plastic
(ii) MSC expansion to provide a cell population of the invention comprising eMSC.

  Optionally, the cell population of the present invention can be cryopreserved during and / or after the expansion step (ii). Optionally, the phenotype of the cell population of the invention can be assessed during and / or after the expansion step (ii). Optionally, the cell population of the invention can be isolated after growth step (ii) and resuspended in a pharmaceutically acceptable carrier and / or diluent.

MSC Isolation MSCs for use in the present invention include, but are not limited to, peripheral blood, bone marrow, placenta, adipose tissue, periosteum, pulp, spleen, pancreas, ligament, skin, tendon, skeletal muscle, umbilical cord and It can be isolated from any suitable tissue such as placenta. In preferred embodiments, MSCs are obtained from connective tissue, such as the interstitial fraction of adipose tissue, hyaline cartilage, bone marrow, skin, and the like.

  In one embodiment, the MSC is derived from the stromal fraction of adipose tissue. In another embodiment, MSCs are obtained from chondrocytes, such as hyaline cartilage. In another embodiment, the MSC is obtained from the skin. In another embodiment, the MSC is obtained from bone marrow.

  MSCs can be obtained from any suitable tissue and from any suitable animal, including humans. Typically, the cells are obtained from mammalian connective tissue after birth.

  MSCs can also be isolated from any organism of the same or different species as the subject. Any organism with MSC can be used in the present invention. In one embodiment, the organism can be a mammal, and in another embodiment, the organism is a human.

  Fat-derived MSCs can be obtained by any means standard in the art. Typically, the cells are obtained by detaching the cells from the source tissue (eg, lipoaspirate or adipose tissue), typically by treating the tissue with a digestive enzyme such as collagenase. The digested tissue material is then filtered through a filter, typically about 20 microns to 1 mm. The cells are then isolated (typically by centrifugation) and cultured on an adherent surface (typically a tissue culture plate or flask). Such methods are known in the art, for example as disclosed in US Pat. No. 6,777,231. According to this methodology, lipoaspirate is obtained from adipose tissue and cells derived therefrom are obtained. In this methodology, cells can be washed, preferably with PBS, to remove contaminating debris and red blood cells. Cells are digested with collagenase in PBS (eg, 30 minutes at 37 ° C., 0.075% collagenase, type I, Invitrogen, Carlsbad, Calif.). To remove residual red blood cells, the digested sample is washed (eg with 10% fetal calf serum), treated with 160 mmol / L ClNH4, and finally with DMEM complete medium (10% FBS, 2 mmol / L glutamine and 1 In DMEM containing% penicillin / streptomycin). Cells can be filtered through a 40 μm nylon mesh.

The cells are cultured in a suitable tissue culture vessel containing a suitable surface for MSC attachment, such as plastic. Non-adherent cells are removed, for example, by washing in a suitable buffer, providing an isolated population of adherent stromal cells (eg, MSCs). Cells isolated by this method are plated on tissue culture flasks (preferably 2-3 × 10 ⁴ cells / cm ² ), and the culture medium is changed every 3-4 days at 37 ° C. and 5% CO _2. Can be grown on. The cells are preferably transferred to a new culture flask (1,000 cells / cm ² ) when the culture reaches 90% confluence.

  Cell isolation is preferably performed under aseptic or GMP conditions.

  In certain embodiments, the cells can be cultured for at least about 15 days, at least about 20 days, at least about 25 days, or at least about 30 days. Typically, the growth of cells in culture improves the phenotypic homogeneity of the cells in the cell population, resulting in a substantially pure or homogeneous population.

  Cells are preferably detached (eg, with trypsin) from the adherent surface when the culture reaches about 75%, 80%, 85%, 90% or 95% confluence and transferred (passaged) to a new culture vessel. )

  In certain embodiments, the cells are passaged at least 3 times, meaning that the cells are expanded in culture with at least 3 passages. In another embodiment, the cells are passaged at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times.

  In certain embodiments, the cells are expanded in culture for at least 3 population doublings. In certain embodiments, the cells are expanded in culture for at least 4, 5, 6, 7, 8, 9, 10, or 15 population doublings. In certain embodiments, the cells are expanded in culture for a population doubling of less than 7, less than 8, less than 9, less than 10, or less than 15. In certain embodiments, the cells are expanded in culture for about 5-10 population doublings.

  The cells can be cultured by any technique known in the art for stromal stem cell culture. A discussion of various culture techniques, as well as their scale-up, can be found in Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, Wiley-Liss 2000. Cells can be expanded using culture flasks or bioreactors suitable for large scale growth. Suitable bioreactors for large scale growth of mesenchymal stromal cells are commercially available and can include both 2D (ie substantially planar) and 3D growth bioreactors. Examples of such bioreactors include, but are not limited to, plug flow bioreactors, reflux bioreactors, continuous stirred tank bioreactors, and stationary-bed bioreactors.

  In certain embodiments, the cells are cultured by monolayer culture. Any medium that can support MSCs in tissue culture can be used. Medium formulations that would support MSC growth include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), α-modified Minimum Essential Medium (αMEM), and Roswell Park Memorial Laboratory Medium 1640 (RPMI Medium 1640). ). Typically, 0-20% fetal bovine serum (FBS) will be added to the medium to support stromal cell growth. However, if growth factors, cytokines, and hormones that are essential in FBS for stromal cells and chondrocytes are identified and provided at appropriate concentrations in the growth medium, a defined medium may be used. It will be possible. Media useful in the methods of the present invention may contain one or more compounds of interest, including but not limited to antibiotics, mitogenic or differentiation-inducing compounds for stromal cells. The cells of the present invention can be grown at temperatures between 31 ° C. and 37 ° C. in a humidified incubator. The carbon dioxide content can be maintained between 2% and 10% and the oxygen content can be maintained between 1% and 22%.

  Antibiotics that can be added to the medium include, but are not limited to, penicillin and streptomycin. The concentration of penicillin in the chemically defined culture medium can be about 10 to about 200 units per ml. The concentration of streptomycin in the chemically defined culture medium can be from about 10 to about 200 μg / ml.

  Typically, the MSC used in the methods of the present invention is an expanded cell population, and preferably the cells are grown to provide a substantially pure or homogeneous population.

  In one embodiment, the cell population is increased until expression of the marker CD34 is reduced compared to freshly isolated non-enhancing cells. For example, increasing the cell population until the expression of the marker CD34 is reduced to a level of less than 50%, less than 35%, less than 30%, or less than 5%, eg 35-5% or 20-10% of the cells in the population Let Typically, the cell population is increased until the expression of the marker CD34 is reduced to a level of less than 5% of the cells in the population. Accordingly, the eMSC population of the invention contains less than 50%, less than 35%, less than 30%, or less than 5% of cells expressing CD34, such as 35-5% or 20-10%.

  In one embodiment, the cell population is increased until expression of the marker STRO-1 is reduced compared to freshly isolated non-enhancing cells. For example, a cell population is reduced until the expression of the marker STRO-1 is reduced to a level of less than 50%, less than 35%, less than 30%, or less than 5% of the cells of the population, eg 35-5% or 20-10% Increase. Typically, the cell population is increased until the expression of the marker STRO-1 is reduced to a level of less than 5% of the cells in the population. Accordingly, the eMSC population of the present invention contains less than 50%, less than 35%, less than 30%, or less than 5% of cells expressing STRO-1, such as 35-5% or 20-10%.

  Typically, the cell population is increased until expression of the markers CD34 and STRO-1 is reduced compared to freshly isolated non-enhancing cells. For example, a cell population is reduced in expression of markers CD34 and STRO-1 to a level of less than 50%, less than 35%, less than 30%, or less than 5%, eg 35-5% or 20-10% of the cells of the population Increase until Typically, the cell population is increased until the expression of the markers CD34 and STRO-1 is reduced to a level of less than 5% of the population's cells. Accordingly, the eMSC population of the present invention contains less than 50%, less than 35%, less than 30%, or less than 5% of cells expressing CD34 and STRO-1, such as 35-5% or 20-10%.

  Increased MSC populations (eg, those in which expression of CD34 and / or STRO-1 is 5% or less) are advantageous because they exhibit lower multipotency than freshly isolated cells. That is, they may have a reduced, reduced or no ability to differentiate into other cell phenotypes compared to naturally occurring non-enhanced MSCs.

  One skilled in the art will appreciate that the methods for preparing the compositions of the present invention are not limiting and compositions of the present invention prepared by any method are within the scope of the present invention. In one embodiment, the invention comprises (a) collecting tissue from a donor; (b) obtaining a cell suspension by enzymatic treatment; (c) precipitating the cell suspension and placing the cells in a culture medium. (D) culturing the cells for at least about 5 days or 5 population doublings, providing a method for preparing a composition of the invention.

  In one embodiment, the cells may be cryopreserved prior to administration, eg, during and / or after growth. Accordingly, the present invention also provides a cryopreserved cell of the present invention. Methods for cryopreserving cells are known in the art and typically require the use of a suitable cryopreservation agent (eg DMSO). The cells can be stored frozen at any point during the isolation and / or growth phase and thawed prior to administration. Typically, cells may be stored frozen at the time of passage 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or more passages. For example, cells can be cryopreserved at 3-10 or 5-10 passages. In some cases, the cells may be re-plated and cultured prior to administration.

  In one embodiment, the cells are isolated from cryopreservation and / or culture medium and resuspended in a pharmaceutically acceptable carrier and / or diluent (eg, DMEM optionally supplemented with serum) prior to administration. be able to.

  In some embodiments, a cell population of the invention in a composition of the invention may be pre-stimulated to increase one or more of its proliferative capacity, mobility, viability, therapeutic effect and immunomodulation. it can. In some embodiments, the pre-stimulation can be achieved by contacting the MSC with a cytokine. In some embodiments of the invention, the pre-stimulation may be achieved by contacting the MSC with IFN-γ.

  In certain embodiments of the invention, MSCs may be pre-stimulated with IFN-γ at a concentration of 0.1-100 ng / ml. In further embodiments, the MSCs are pre-stimulated using IFN-γ concentrations of 0.5-85 ng / ml, 1-70 ng / ml, 1.5-50 ng / ml, 2.5-40 ng / ml, or 3-30 ng / ml. can do. Pre-stimulation can be performed by stimulation for about 12 hours or more. Pre-stimulation can be performed by stimulation for about 13 hours or more, about 18 hours or more, about 24 hours or more, about 48 hours or more, or about 72 hours or more.

  In one embodiment, the MSCs of the invention can be stably or transiently transfected or transduced with the nucleic acid of interest using a plasmid, virus or other vector strategy. Nucleic acids of interest include, but are not limited to, those that encode a gene product that increases the production of the extracellular matrix component found in the tissue type to be repaired, eg, the intestinal or vaginal wall.

  Introduction of a viral vector having a regulatory gene into a stromal cell is not limited, but is purified at a multiplicity of infection (virus unit: cell) of about 10: 1 to 2000: 1 (eg, by the cesium chloride band method) Can be carried out using viral vectors containing adenoviruses, retroviruses or adeno-associated viruses. Cells can be exposed to the virus for about 1 to about 24 hours in serum-free or serum-containing media in the absence or presence of cationic surfactants such as polyethyleneimine or Lipofectamine ™ (Byk T. et al. (1998) Human Gene Therapy 9: 2493-2502; Sommer B. et al. (1999) Calcif. Tissue Int. 64: 45-49).

  Other suitable methods for introducing vectors or plasmids into stromal cells include, for example, U.S. Pat.Nos. 5,578,475; 5,627,175; 5,705,308; 5,744,335; 5,976,567; 6,020,202; and 6,051,429. And lipid / DNA complexes such as those described. Suitable reagents include lipofectamine, polycationic lipid 2,3-dioleyloxy-N- [2- (sperminecarboxy-amido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA ) (Chemical abstract registration name: N- [2- (2,5-bis [(3-aminopropyl) amino] -1-oxypentyl} amino) ethyl] -N, N-dimethyl-2,3-bis ( 9-octadecenyloxy) -1-propanamine-ium trifluoroacetate) and neutral lipid dioleoylphosphatidylethanolamine (DOPE) in water filtered through a membrane filter 3: 1 (w / w) Liposomal preparations can be mentioned. An example is the Lipofectamine 2000 ™ formula (available from Gibco / Life Technologies, # 11668019). Other reagents include FuGENETM 6 transfection reagent (mixture of non-liposomal lipids and other compounds in 80% ethanol, available from Roche Diagnostics Corp., # 1814443), and LipoTAXI ™ transfection reagent (Invitrogen Corp. Lipid formulation from # 204110). Transfection of stromal cells can be performed by electroporation as described, for example, in M.L. Roach and J.D. McNeish (2002) Methods in Mol. Biol. 185: 1. Suitable viral vector systems for the production of stromal cells with stable genetic changes may be based on adenoviruses and retroviruses and can be prepared using commercially available viral components.

  Transfection of plasmid vectors with regulatory genes into MSCs can be accomplished in monolayer cultures using calcium phosphate DNA precipitation or the use of cationic detergent methods (Lipofectamine ™, DOTAP), or in plasmid DNA in biocompatible polymers. It can be achieved in three-dimensional culture by directly integrating the vector (Bonadio J. et al. (1999) Nat. Med. 5: 753-759).

  For the tracking and detection of functional proteins encoded by these genes, viral or plasmid DNA vectors are easily detectable marker genes, such as green fluorescent protein or β-galactosidase, both of which can be tracked by histochemical means. Enzymes can be included.

  Following growth, the cell population of the invention is preferably assayed to determine the expression of characteristic markers to confirm its phenotype, which can be performed using traditional means. .

  The term “expression” is used to describe the presence of a marker in a cell. In order to be considered expressed, the marker must be present at a detectable level. “Detectable level” means that the marker can be detected using one of standard laboratory techniques such as PCR, blotting or FACS analysis. Analysis of phenotypic surface markers of MSC populations can be performed by any method known in the art. By way of example and not limitation, this phenotypic analysis can be performed by staining individual cells. Such staining can be achieved through the use of antibodies. This may be direct staining using a labeled antibody or indirect staining using a second labeled antibody against a primary antibody specific for the cell marker. Antibody binding can be detected by any method known in the art. Antibody binding can also be detected by flow cytometry, immunofluorescence microscopy or radiography.

  Alternatively, or additionally, the gene corresponds to an intracellular expression level of at least about 100 copies per cell and can be expressed by the cells of the population of the invention if expression can be reasonably detected after 30 PCR cycles. Is considered to be. The terms “express” and “expression” have corresponding meanings. At expression levels below this threshold, the marker is considered not expressed. Comparison of the expression level of one marker in adult stromal cells of the present invention with the expression level of the same marker in another cell, such as an embryonic stem cell, preferably compares two cell types isolated from the same species Can be implemented. Preferably this species is a mammal, more preferably this species is human. Such comparisons can be conveniently performed using reverse transcriptase polymerase chain reaction (RT-PCR) experiments.

  Cell surface markers can be identified by any suitable traditional technique, usually based on positive / negative selection. For example, monoclonal antibodies against cell surface markers to be confirmed for their presence / absence in cells can be used, but other techniques can also be used. Thus, in certain embodiments, CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105 are one, two, three, four, five, six, Monoclonal antibodies against species, 7 or all are used to confirm the absence of the marker in selected cells. In addition, factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133 1, 2, 3, 4 Monoclonal antibodies against species, or all, are used to confirm the presence or detectable expression level of at least one, preferably all, of the above markers.

  In further embodiments, at least one, two, three or all of CD34, CD45, CD31, CD14, eg, monoclonal antibodies against CD31 and / or CD34 confirm the presence or detectable expression level of the marker. Used for. In a further embodiment, monoclonal antibodies against CD34 are used to confirm the absence of the marker in selected cells. In a further embodiment, monoclonal antibodies against STRO-1 are used to confirm the absence of the marker in selected cells. In a further embodiment, monoclonal antibodies against at least one, two, three or all of CD29, CD59, CD90, CD105, e.g. CD59 and / or CD90, confirm their presence or detectable expression level. Used for.

  Such monoclonal antibodies are known and are commercially available or can be obtained by those skilled in the art by traditional methods.

IFN-γ induced IDO activity in selected cells can be determined by any suitable traditional assay. For example, selected cells can be stimulated with IFN-γ and assayed for IDO expression, followed by traditional Western blot analysis for IDO protein expression, and IFN-γ of selected cells. IDO enzyme activity after stimulation can be measured, for example, by conversion of tryptophan to kynurenine via high performance liquid chromatography (HPLC) analysis and photometric determination of kynurenine concentration in the supernatant as readout. Since the cells of the invention express IDO under certain conditions, any suitable technique that allows detection of IDO activity after IFN-γ stimulation can be used to select the cells of the invention. . A suitable assay for determining IFN-γ induced IDO activity in selected cells is disclosed in WO 2007/039150. The amount of IDO produced depends on the number of cells per square centimeter, which is preferably at a level of 5000 cells / cm ² or higher, but is not limited to this concentration, and the concentration of IFN-γ is ideally Although it is 3 ng / ml or more, it is not limited to this concentration. The activity of IDO produced under the conditions described will result in detectable production of kynurenine in the μM range after more than 24 hours.

In one embodiment of administration , the composition of the invention may be prepared for systemic administration (eg, rectal, nasal, buccal, intravaginal, via implanted reservoir, or via inhalation). In another embodiment, the composition of the present invention may be prepared for topical administration. The composition of the present invention may be administered by a parenteral route. The composition may be administered by subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, intralymphatic and intracranial routes. In one embodiment, the MSC is administered by an intravenous route, such as by intravenous infusion. Alternatively, MSCs can be passed through an intralymphatic route, eg, by intralymphatic injection, to, for example, but not limited to lymph nodes, most preferably peripheral organs including peripheral or inguinal lymph nodes. Administered by intralymphatic injection. In each of these embodiments, the MSC can be ASC, eg, eASC. As used herein, the term “lymphatic system” should be given its ordinary meaning in the art and refers to lymphoid tissues such as lymphoid organs connected by the conduction system of lymphatic vessels and capillaries. . The term “lymphoid organ” refers to a lymph node, most preferably an axilla or inguinal lymph node.

  Typically, the patient is a human.

  Accordingly, in one embodiment, the present invention provides rheumatoid arthritis in a human subject, preferably moderate rheumatoid arthritis (CDAI score> 10 to ≦ 22, and optionally DAS28 score> 3.2 to ≦ 5.1 and / or RAPID3 score> ASC for use in the treatment of 6 to ≦ 12), wherein the ASC is administered intralymphaticly, for example by intralymphatic administration. In another embodiment, the present invention relates to rheumatoid arthritis in a human subject, preferably moderate rheumatoid arthritis (CDAI score> 10 to ≦ 22, and optionally DAS28 score> 3.2 to ≦ 5.1 and / or RAPID3 score> 6. ~ <12) for providing an ASC for use, wherein the ASC is administered intravenously, for example by intravenous infusion. ASC can optionally be administered repeatedly at least one day apart.

  In one embodiment, the MSC used in the present invention may be self with respect to the subject being treated. In further embodiments, MSCs used in the present invention can be homologous or xenogeneic to the subject being treated. Donor-derived allogeneic MSCs can theoretically be used for the treatment of any patient, despite MHC incompatibility. In one embodiment, the compositions of the invention can be administered by injecting or implanting the composition into one or more target sites of the subject to be treated. In further embodiments, the compositions of the invention may be inserted into a delivery device that facilitates introduction of the composition into a subject by injection or implantation. In one embodiment, the delivery device can include a catheter. In further embodiments, the delivery device may include a syringe.

Dose
The dosage of MSC and any additional therapeutic agent will vary depending on the patient's symptoms, age and weight, nature and severity of the disease to be treated or prevented, route of administration, and form of additional therapeutic agent. The compositions of the invention can be administered in single or divided doses. Appropriate doses of MSC and any additional therapeutic agents can be determined by known techniques.

Typically, the dosage is about 10 × 10 ⁶ cells or less per kg body weight of the subject, about 9 × 10 ⁶ cells or less per kg, about 8 × 10 ⁶ cells or less per kg, and about 7 × 10 ⁶ per kg. ⁶ cells or less, about 6 × 10 ⁶ cells or less per kg, and about 5 × 10 ⁶ cells or less per kg. In another embodiment, the dosage is about 0.25 × 10 ⁶ cells / kg to about 5 × 10 ⁶ cells / kg; or more preferably about 1 × 10 ⁶ cells / kg to about 5 × 10 ⁶ cells / kg Can be between. Thus, in another further embodiment, the dosage is about 0.25 × 10 ⁶ cells / kg, about 0.5 × 10 ⁶ cells / kg, about 0.6 × 10 ⁶ cells / kg, about 0.7 × 10 ⁶ cells / kg, About 0.8 × 10 ⁶ cells / kg, about 0.9 × 10 ⁶ cells / kg, about 1.1 × 10 ⁶ cells / kg, about 1.2 × 10 ⁶ cells / kg, about 1.3 × 10 ⁶ cells / kg, about 1.4 × 10 ⁶ Cells / kg, about 1.5 × 10 ⁶ cells / kg, about 1.6 × 10 ⁶ cells / kg, about 1.7 × 10 ⁶ cells / kg, about 1.8 × 10 ⁶ cells / kg, about 1.9 × 10 ⁶ cells / kg, or It can be about 2 × 10 ⁶ cells / kg. In another embodiment, the dosage is between 0.1 and 1 million cells / kg, between 1 and 2 million cells / kg, between 2 and 3 million cells / kg, between 3 and 4 million cells / kg. kg, 4-5 million cells / kg, 5-6 million cells / kg, 6-7 million cells / kg, 7-8 million cells / kg, 8-8 It can be between 9 million cells / kg, or between 9-10 million cells / kg.

In another embodiment, the cells may be administered to the patient at a fixed dose regardless of the patient's weight. Typically, the dosage is between about 10 million cells to 500 million cells, for example, the dosage is about 10 × 10 ⁶ cells, 50 × 10 ⁶ cells, 10 × 10 ⁷ cells, 50 × 10 ⁷ cells.

  In one embodiment, the invention provides an MSC, such as ASC, for use in the treatment of rheumatoid arthritis in a human subject, such as moderate rheumatoid arthritis, wherein the MSC is 0.05 per kg body weight of the subject. Administered by intralymphatic route, for example by intralymphatic infusion, at doses between 0.1 and 1 million cells per kg body weight of the subject, between ˜0.25 million cells. In another embodiment, a fixed dose of between about 0.1-5 million cells, or about 5 million cells, or about 10 million cells is administered by the intralymphatic route, for example by intralymphatic infusion.

In another embodiment, the invention provides an MSC, e.g., ASC, for use in the treatment of rheumatoid arthritis, e.g., moderate rheumatoid arthritis, in a human subject, wherein the MSC is per kg body weight of the subject. Administered by intravenous route, such as intravenous infusion, at a dosage of about 0.25 × 10 ⁶ cells to about 10 × 10 ⁶ cells, for example about 0.25 × 10 ⁶ cells to about 5 × 10 ⁶ cells per kg body weight of the subject Is done. A typical dosage is 4 × 10 ⁶ cells per kg subject body weight. 8 × 10 ⁶ cells per kg body weight of the subject can also be used.

  The exact time of administration and the amount of any particular drug that results in the most effective treatment in a given patient depends on the activity, pharmacokinetics, and bioavailability of that drug, the patient's physiological condition (age, gender, disease Type and stage, general physical condition, predetermined dose and responsiveness to the type of drug), route of administration, etc. The information presented herein may be used to optimize treatment times, such as optimal time of administration and / or the extent of routine experimentation such as subject monitoring and dose and / or timing adjustment. Can be used for dose determination. While the subject is being treated, the health status of the subject can be monitored by measuring one or more relevant indicators at predetermined time points during a 24 hour period. Treatment regimes such as dosage, administration time and formulation can be optimized according to the results of such monitoring.

  Treatment can be initiated from smaller doses that are less than the optimum dose. Thereafter, the dosage can be increased by small increments until the optimum therapeutic effect is achieved.

  Since the onset and duration of the effects of the different components can be complementary, the combination of several therapeutic agents can reduce the dosage required for the individual components. In such combination therapy, the different active agents can be delivered together or separately and simultaneously or at different times of the day.

Further therapeutic agents In one embodiment, the pharmaceutical composition of the invention comprises one or more (or two or more, or three or more, for example one, two, three, four or five). It may contain a therapeutic agent or may be administered in combination with a further therapeutic agent.

  In some embodiments, the MSC and one or more additional therapeutic agents may be administered to the subject simultaneously. In other embodiments, the MSC and one or more additional therapeutic agents may be administered sequentially to the subject. One or more additional therapeutic agents may be administered before or after administration of MSC.

  Said further therapeutic agent may be selected from: analgesics such as non-steroidal anti-inflammatory agents, opiate agonists or salicylates; anti-infectives such as anthelmintics, anti-anaerobic fungi, antibiotics, aminoglycosides Antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, β-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, Antimycobacterial drugs, antituberculous antimycobacterial drugs, antiprotozoal drugs, antimalarial antiprotozoal drugs, antiviral drugs, antiretroviral drugs, anti-scabies drugs, anti-inflammatory drugs, corticosteroid anti-inflammatory drugs, anti-pruritic drugs / topical Anesthetics, local anti-infectives, antifungal local anti-infectives, antiviral local anti-infectives; electrolysis and renal agents, eg acids Agents, alkalizing agents, diuretics, carbonic anhydrase inhibitors diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte replacement, and uric acid excretion drugs; enzymes such as pancreas Enzymes and thrombolytic enzymes; gastrointestinal drugs such as antidiarrheal drugs, antiemetics, gastrointestinal anti-inflammatory drugs, salicylate gastrointestinal anti-inflammatory drugs, antacid anti-ulcer drugs, gastric acid-pump inhibitory anti-ulcer drugs, gastric mucosal anti-ulcers Drugs, H2 blocker anti-ulcer drugs, cholelitholytic agents, digestive drugs, vomiting drugs, constipation and stool softeners, exercise promotors; general anesthetics such as inhalation anesthetics, halogenated inhalation anesthetics, intravenous Anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics, and opiate agonist intravenous anesthetics; hormones or hormone regulators such as abortion drugs, adrenal agents, corti Steroid adrenal agonists, androgens, antiandrogens; immunobiological agents such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics such as amide local anesthetics and ester local anesthetics; musculoskeletal agents (Musculoskeletal agent), eg anti-gout anti-inflammatory agent, corticosteroid anti-inflammatory agent, gold compound anti-inflammatory agent, immunosuppressive anti-inflammatory agent, non-steroidal anti-inflammatory agent (NSAID), salicylic acid anti-inflammatory agent, mineral And vitamins such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

  The additional therapeutic agent preferably comprises one or more of a disease modifying anti-rheumatic drug (DMARD), a non-steroidal anti-inflammatory drug (NSAID), a biopharmaceutical and a corticosteroid. NSAIDs include, but are not limited to, Cox-2 inhibitors, diclofenac, ibuprofen, naproxen, celecoxib, mefenamic acid, etlicoxib, indomethacin, and aspirin. Corticosteroids include, but are not limited to, triamcinolone, cortisone, prednisone, and methylprednisolone. Disease modifying anti-rheumatic drugs (DMARDs) include, but are not limited to, auranofin, chloroquine, cyclosporine, cyclophosphamide, gold preparation, hydroxychloroquine sulfate, leflunomide, methotrexate, penicillamine, sodium aurothiomaleate, Sulfasalazine is mentioned.

  In one embodiment, the patient preferably has at least two or three DMARDs selected from the group consisting of hydroxychloroquine, leflunomide, methotrexate, minocycline, and sulfasalazine (eg, methotrexate and hydroxychloroquine; methotrexate and leflunomide; methotrexate and sulfasalazine). Sulfasalazine and hydroxychloroquine; methotrexate, hydroxychloroquine and sulfasalazine).

  In some cases, the patient can be further treated with a biopharmaceutical. Biopharmaceuticals include, but are not limited to, selective costimulatory modulators, Tnf-a inhibitors, IL-1 inhibitors, IL-6 inhibitors, and B-cell targeted therapeutic agents. Typically, the patient can be further treated with at least one biological therapy, such as a TNF-α inhibitor. Thus, in one embodiment of the invention, the patient further comprises a TNF-α inhibitor, typically adalimumab (Fumila), certolizumab (Simdia), etanercept (Enbrel), golimumab (Simponi), and / or infliximab (Remicade) Can be treated with at least one biological therapy.

  In another embodiment, the additional therapeutic agent can be a growth factor or other molecule that affects cell growth or activation. In further embodiments, growth factors can induce terminal differentiation. In another embodiment, the growth factor can be a variant or fragment of a natural growth factor. Methods for producing such variants are well known in the art and can include, for example, making conservative amino acid changes, or mutagenesis and assays for the required functionality of the resulting variant.

  The invention is further illustrated by the following examples. These examples are provided for illustrative purposes only, and are not intended to be limiting.

Example 1: Initial treatment with ASC reduces the exacerbation of arthritis On the day of the study, the tail (from the trunk) of each mouse (a young healthy 8 week old male DBA / 1 (H-2 ^q ) mouse) 2-3 cm) was subcutaneously injected with a dose of type II chicken collagen emulsion (final concentration 1 mg / ml) in complete Freund's adjuvant (final concentration 2 mg / ml of Mycobacterium tuberculosis) at a volume of 0.1 ml / animal .

  The distribution of animals to each experimental group was randomized at the start of the study. Treatment was carried out 15 days, 18 days and 21 days after collagen injection (n = 12 mice per group). The arthritis score was monitored from day 21. Adipose tissue-derived stem cells (ASC) were obtained from human lipoaspirate (see Example 7) and administered as a suspension in lactated Ringer's solution (vehicle) via the intravenous route (tail vein) (200,000 cells).

The arthritic index score was evaluated for each animal from day 21 (from collagen injection) to the end of the study. The severity of arthritis was scored for both fore and hind legs according to the following arthritis index scoring system. The final score is the sum of the four foot scores. The maximum score is 16.

Experimental group:
A) CIA, no treatment (n = 12)
B) Intravenous injection of ASC into CIA, d15, d18, and d21 (200,000 cells) (n = 12)
As shown in FIG. 1, treatment of mice with early arthritis with ASC significantly reduced the disease course (FIG. 1A) and incidence (FIG. 1B).

Example 2: Long-term treatment with ASC prolongs the therapeutic effect in early arthritis On the day of the study, the tail of each mouse (a young healthy 8-week-old male DBA / 1 (H-2 ^q ) mouse) 0.1 ml / animal volume of the first dose of type II chicken collagen emulsion (final concentration 1 mg / ml) in complete Freund's adjuvant (final tuberculosis tuberculosis 1 mg / ml) in complete Freund's adjuvant (final concentration 1 mg / ml) Was injected subcutaneously. Then, 21 days after the first injection of collagen, a second injection (booster) of type II collagen (0.1 ml / animal) was subcutaneously administered to each animal again at the tail but at a different position from the first injection. . At this time, a collagen suspension was prepared using incomplete Freund's adjuvant (without Mycobacterium tuberculosis).

  In this study, treatment with ASC began when the early stages of arthritis were already established (arthritic index score 2-4). In order to ensure uniformity of the arthritis index scores in each experimental group, animals were not distributed to each experimental group in a random manner at the start of the study. Instead, animals that reached an arthritis index score between 2-4 were assigned to the experimental group (n = 12 per group). The arthritis score was monitored as described above.

Experimental group:
A) CIA, no treatment (n = 12)
B) Intravenous injection of ASC into CIA, d1, d3, d5 (200,000 cells) (n = 12)
C) ASC injected intravenously into CIA, d1, d3, d5, d8, d15, d22, d29 (200,000 cells) (n = 12)
As shown in FIG. 2, treatment with ASC at d1, d3 and d5 reduced the severity of the disease. In particular, long-term treatments (d8, d15, d22 and d29) increased and prolonged the therapeutic effect of the cells.

Example 3: Optimization of dose and timing of ASC treatment for early arthritis Experimental group
A) Healthy (n = 5)
B) CIA, no treatment (n = 10)
C) ASC cultured in CIA, d1, d3, d5 is injected intravenously (1,000,000 cells) (n = 11)
D) ASC cultured in CIA, d1, d3, d5 injected intravenously (200,000 cells) (n = 11)
E) ASC cultured in CIA, d1, d3, d5 injected intravenously (40,000 cells) (n = 11)
F) ASC cultured in CIA, d1, d3, d5 is injected intravenously (8,000 cells) (n = 11)
G) ASC cultured in CIA, d1, d8, d15 is injected intravenously (1,000,000 cells) (n = 11)
H) Intravenous injection of ASC cultured in CIA, d1, d8, d15 (200,000 cells) (n = 11)
I) ASC cultured in CIA, d1, d8, d15 is injected intravenously (40,000 cells) (n = 11)
J) ASC cultured in CIA, d1, d8, d15 is injected intravenously (8,000 cells) (n = 11)

Young healthy 8-week-old male DBA / 1 (H-2 ^q ) mice were used in this study. CIA was generated as shown in Example 2 and treatment was initiated after the mice showed an early stage of the disease (arthritis score 2-4).

  As shown in Figs. 3 and 4, three veins of ASC are given at d1, d3 and d5 (Fig. 3) at one day intervals, or at d1, d8 and d15 at one week intervals (Fig. 4). When administered internally, the severity of arthritis was reduced. A dose of 1 million to 0.04 million cells was particularly effective.

Example 4: Treatment with ASC reduces pro-inflammatory cytokines in serum and increases T cells with regulatory phenotype in peripheral blood (Days 1, 8 and On day 15, mice were treated intravenously with 200,000 ASCs), peripheral blood was collected, pro-inflammatory cytokines were determined in plasma, and regulatory T cell populations were measured in cell fractions in blood. As shown in FIG. 5, treatment with ASC decreased the levels of IL6 (FIG. 5A), IL17 (FIG. 5B) and IL23 (FIG. 5C) and increased the level of regulatory T cells (FIG. 5D). Furthermore, treatment with ASC, as determined by a significant decrease in bone (Fig. 6A) and trabecular (Fig. 6B) mineral density loss compared to healthy mice 50 days after the start of treatment (micro CT scanning). ), Reduced joint damage mediated by arthritis.

Example 5: Intralymphatic administration of ASC in the early stages of arthritis tested young healthy 8-week-old male DBA / 1 (H-2 ^q ) mice that reduce arthritic score, foot edema and joint damage used. CIA was generated as shown in Example 2 and treatment was initiated after the mice showed an early stage of the disease (arthritis score 2-4).

Experimental group:
A) CIA, no treatment (n = 12)
B) Intralymphatic treatment of ASC at CIA, d1, d8, and d15 (48,500 cells in each of the right and left inguinal lymph nodes) (n = 12)

  In addition to the arthritis score, hind paw edema was monitored (using a plethysmometer). As shown in FIG. 8, intralymphatic administration of ASC in the early stages of arthritis caused disease exacerbation as shown by a decrease in the group's mean arthritis score (FIGS. 8A and B) and paw volume (FIGS. 8C and D) Was suppressed. The average arthritis score is the average clinical score across the study.

  Furthermore, as shown in FIG. 7, joint damage (loss of bone and trabecular mineral density compared to healthy mice) on the 50th day was reduced by administration of ASC into the lymphatic vessels.

Example 6: The effect of ASC administered into lymphatic vessels is mediated by induction of T cells with a regulatory phenotype (CD4 + CD25 + FoxP3 +) and T cells with an anti-inflammatory phenotype (CD4 + IL10 + cells) Arthritis (CIA) was induced and mice were treated intralymphatically with ASC as described in Example 5. Twenty-two days after the start of treatment, mice were sacrificed and the levels of CD4 + CD25 + T cells expressing FoxP3 (regulatory T cells) and CD4 T cells expressing the anti-inflammatory cytokine IL10 in the spleen and lymph nodes were measured. As shown in FIG. 9, the level of FoxP3 expressing CD4 + CD25 + T cells is elevated in the spleen (FIG. 9A) and lymph nodes (FIG. 9B) of ASC-treated mice compared to untreated mice. As shown in FIG. 10, the level of CD4 T cells expressing IL10 is also elevated in the spleen (FIG. 10A) and lymph nodes (FIG. 10B) of ASC-treated mice compared to untreated mice.

  These examples show that treatment with ASC within a particular time frame (early stage of disease when the disease is moderate) is surprisingly effective. Particularly good results can be achieved when ASCs are administered at intervals and / or via the intralymphatic route.

Example 7: Administration of ASC is particularly effective in human patients with early / moderate rheumatoid arthritis This study is a multicenter, dose-escalated, randomized, single-blind, placebo-controlled phase Ib / IIa clinical trial, A follow-up period of up to one month was established (EudraCT no .: 2010-021602-37; clinicaltrials.gov number: NCT01663116).

  The objective was to evaluate the safety and tolerability of intravenous allogeneic increased adipose-derived stem cells (eASC) in patients with refractory rheumatoid arthritis (RA), as well as preliminary clinical studies in this population It was to acquire activity data.

eASC
The lipoaspirate was digested with collagenase after extensive washing. The erythrocytes were then removed by hemolysis and a stromal vascular fraction was obtained by filtration. ASCs were isolated and in vitro amplification was performed by sequential amplifying passages. Finally, eASC was subjected to a cryopreservation process to obtain a master cell bank, from which a new amplification process was performed to obtain a working cell bank, which was also cryopreserved. Batches for clinical use were obtained after thawing the cells from the working cell bank, harvesting the cells at the appropriate culture conditions and formulating it into the corresponding dosing vehicle. At various stages of the process, eASCs were tested for cell number, viability, population doubling, morphology, potency, identity, purity, sterility, mycoplasma and genetic stability in quality control. Products for clinical use were released after confirming compliance with established quality standards.

Because there was no safety and efficacy data for intravenous administration of eASC or other mesenchymal stem cells to RA patients at the time of patient study design, the sample size was determined for other mesenchymal stem cells for other applications. Based on clinical trials.

  Eligible patients are adults who have been diagnosed with RA for more than 6 months, have been treated with at least one non-biological agent, and have previously failed treatment with at least two biologics. did. Their EULAR disease activity score (DAS28-ESR) must be> 3.2 and has 4 tender joints and 4 swollen joints (based on 68/66 joints) for palpation You must be treated as an outpatient.

  A total of 67 patients were enrolled at 18 testing institutions. Fourteen patients were screened out, so 53 patients continued the trial and received at least one dose of study treatment (ITT population). The PP population consists of 48 patients because 5 patients had major protocol violations. Ten patients discontinued the study early (4 in Cohort A, 1 in Cohort B, 2 in Cohort C, 3 in the placebo group).

  Randomize 53 patients with active refractory RA (refractory to at least 2 biologics), 1 million on day 1, 8 and 15: / kg (Cohort A; 20 people), 2 million / kg (Cohort B; 6 people), 4 million / kg (Cohort C; 6 people), or placebo (7 people) 3 times intravenously Administration was by infusion (iv) and followed for 24 weeks for treatment evaluation. Background non-biological DMARDs and NSAIDs and glucocorticoids (≦ 10 mg / day prednisone or equivalent) remained stable. Relief treatment with any DMARD, including biologics, was possible after 3 months. Visits for follow-up are week 1, 2 and 3 (visit 1-3), and 1 month, 2 months, 3 months, 4 months, 5 months and 6 months (visit 4-9) Carried out. In order to keep patient safety as much as possible, safety assessments were single blind (patient blind, patient open label), while efficacy assessment was double blind. This study was conducted according to the applicable laws and regulations in accordance with the GCP standard (CPMP / ICH / 135/95) and the revised Declaration of Helsinki (Seoul, October 2008) after obtaining approval from the corresponding ethics committee. Written informed consent was obtained from the patient.

  Baseline demographic and clinical characteristics are typical of refractory RA, and are generally higher among treatment groups (the number of previous DMARDs was higher in Cohort C compared to other groups). Except). The average (SD) number of previous biologics was 2.92 (1.44).

Statistical analysis Descriptive analysis of anthropometric data, variables related to the patient's medical history, efficacy endpoints reported at baseline, and baseline laboratory parameters were performed. Analysis of primary safety endpoints was performed on the treatment intention (ITT) population. The number and percentage of patients who experienced AEs, SAEs, treatment-related AEs, and treatment-related SAEs were described for the entire population, by treatment group, and for patients who reported grade 3-4 AEs. These values may be compared between groups in the maintenance phase using the χ ² test. Alternatively, Fisher's exact test was used. Laboratory parameters, especially those included in the selection criteria, are listed for each visit.

A second efficacy endpoint analysis was performed on the ITT and protocol-per-protocol (PP) population. ACR and EULAR responses at 24 weeks were compared between groups using the Cochran-Mantel-Haenszel χ ² test. The primary efficacy endpoints between treatment groups were compared using analysis of variance (ANCOVA), which included the base value in the model as a covariate. Statistical analysis was performed using SAS package v9.2.

Clinical efficacy
・ Moderate RA
Patients were divided into a population with moderate rheumatoid arthritis at baseline (DAS28 (PCR) values 3.2-4.5) and a population with severe rheumatoid arthritis (DAS28 (PCR) values greater than 5.5). In the moderate RA population, patient exacerbations after 3 intravenous doses of eASC were suppressed to levels below 4 and were controlled for 6 months after treatment. However, in the group of patients with severe rheumatoid arthritis, disease suppression is less controlled at a later stage after the fifth visit (FIG. 11A).

  FIG. 11B shows the normalized DAS28 value when the baseline is 100%. The data show that patients with low levels of DAS28 have a lower decrease in DAS28 levels during the first 3 months compared to the group of patients with high levels of DAS28.

  In the case of CDAI scores, patients with low CDAI scores maintained low scores over multiple visits, while patients with high scores did not reach low levels. FIG. 12A shows a group of 8 patients with a CDAI score of 22 or less at baseline (BS) and 6 months of treatment (visit 4, 5, 6, 7, 8, and 9), and baseline and treatment 6 months. Shown is the average of a group of 26 patients with CDAI scores of 22-40 (after visits 4, 5, 6, 7, 8, and 9).

  FIG. 12B shows numbers normalized by regarding the baseline as a 100% score. The data show that patients with low levels of CDAI (<22 at baseline) show significantly greater CDAI levels compared to patient groups with high CDAI levels (> 22 and <40 at baseline). It is.

ACR20 / 50/70 response The second preliminary efficacy endpoint included the proportion of ACR20, ACR50 and ACR70 patients, and the EULAR response (DAS28-ESR and DAS28-CRP). Other efficacy results considered were the Short Form 36 Health Survey (SF-36) questionnaire and the RA MRI score (RAMRIS).

  Patients treated with pooled eASC were compared experimentally with patients treated with placebo. In the ITT population, ACR20, ACR50 and ACR70 were achieved in a higher proportion of patients treated with eASC compared to those treated with placebo (FIGS. 13A, B and C, respectively). Results in ITT and PP populations were similar. Evaluating efficacy in the ITT population according to EULAR criteria also tended to have a better clinical course in patients treated with eASC compared to those treated with placebo. FIG. 14A shows the percentage of patients who achieved a good response according to the EULAR response (DAS28-ESR shows a score of ≦ 3.2 and an improvement of> 1.2), and FIGS. 14B and 14C respectively show patients with low disease activity (DAS28 -ESR <3.2) and patients showing clinical remission (DAS28-ESR <2.6). Of note, in contrast to the pooled eASC group, patients in the placebo group did not show a good EULAR response, low disease activity or clinical remission at any time point. The improvement in DAS28-ESR from baseline was maintained in patients treated with eASC, while the placebo group showed variable response (FIG. 14D). The result of DAS28-CRP was similar to the result of DAS28-ESR. The EULAR response in the PP population was not different from that obtained in the ITT population.

  Overall, the proportion of clinical responses assessed by ACR score was higher in patients treated with eASC than those treated with placebo. Evidence of efficacy is also seen using results such as good EULAR response, low disease activity, or clinical remission, as opposed to those that occurred in the placebo group (no responders at any time). It was. Patients treated with eASC showed sustained improvement in DAS28-ESR over time from baseline.

Conclusion : Evaluation of clinical efficacy showed a trend toward a better clinical course in the group of patients treated with eASC compared to the group treated with placebo. ASC treatment is particularly effective in patients with moderate rheumatoid arthritis, defined as having a DAS28 level of 4.5 or less and a CDAI level of 22 or less even after failure with a biologic. A subset of patients with low CDAI levels (22 and below) showed the best response to ASC treatment compared to subsets with high CDAI levels (22-40).

  Thus, ASC treatment has a moderate rheumatoid arthritis patient with a DAS28 score> 3.2 to ≦ 5.1, a CDAI score> 10 to ≦ 22 and / or a RAPID3 score> 6 to ≦ 12, especially a CDAI score> 10 to ≦ 22 It is particularly useful for the person.

safety
Intravenous infusion of eASC was generally well tolerated with no apparent dose-related toxicity in the dose range tested. The intensity of AEs was evaluated in the range of 1 to 5 according to the National Cancer Institute “Common Terminology Criteria for AEs” 4.0th edition (National Cancer Institute NIoH, US Department of Health and Human Services, Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0. 2010 [cited in 2014]; http://evs.nci.nih.gov/ftp1/ CTCAE / CTCAE_4.03_2010-06- 14_QuickReference_5x7.pdf) A total of 141 adverse events (AEs) were reported, 41 of which were considered treatment-related. At least one AE occurred in 17 patients (85%) in Group A, 15 patients in Group B (75%), 6 patients in Group C (100%), and 4 patients in the placebo group (57%). Two treatment-related severe AEs (one in Cohort A, one in the placebo group) and one treatment-related severe AE (SAE) were reported (Cohort A). The most common AEs (≥5%) were fever (9 cases, 17%), respiratory infection (8 cases, 15%), headache (6 cases, 11%), urinary tract infections (6 cases, 11%) %), Nausea (5 cases, 9%), joint pain (3 cases, 6%), asthenia (3 cases, 6%), malaise (3 cases, 6%) and vomiting (3 cases, 6%) The most common treatment-related AEs were fever (7 cases, 13%), headache (4 cases, 8%), and asthenia (3 cases, 6%). Systemically, the most frequently reported AE was infection. No opportunistic infections were reported except for 1 case of vulvovaginal candidiasis and 1 case of mild herpes simplex virus infection. Four infections were considered to be associated with study treatment: two moderate respiratory infections in Cohort A, and one mild urinary tract infection and one mild Cohort C Stye. Of the 41 treatment-related AEs, 2 were grade 3 strength (severe), 1 was cohort A (lacuna infarction), and 1 was placebo (asthenia). Three SAEs were reported and all occurred in eASC-treated patients: one with lacunar infarction, one with radial nerve palsy, and one with fever. Only lacunar infarcts were suspected to be treatment related. Lacunar infarction was considered a DLT according to previously established definitions. This event includes 3 consecutive SAEs (2 generalized weaknesses and 1 left unilateral dull sensation and 1 paralytic ataxic gait, finally diagnosed with lacunar infarction). The appearance of these symptoms was temporary and the patient recovered without sequelae. This was the only patient who discontinued the study due to AE. No abnormal experimental values were reported other than those related to the reported AEs, and no associated vital sign abnormalities occurred. No malignancy or death was reported.

  Intravenous treatment with allogeneic eASCs generally appears to be well tolerated. 83% of eASC-treated patients experienced some adverse events, most of which were irrelevant to study treatment (71%) and of mild or moderate intensity (94%). No life threatening event (grade 4) or death occurred.

  Interestingly, there was no clear relationship between dose and tolerability. In practice, the proportion of treatment-related AEs was lower in Cohort C than in both Cohorts A and B, which is probably related to the smaller size of Cohort C. In addition, because of the potential initial stress associated with iv infusion of stem cells, the likelihood of overreporting AEs in the lower-dose cohort is associated with the open-label nature of safety trials. Can occur (trial started with low dose administration). This suggests that the safety profile of the maximum dose level evaluated can be similar to the lower dose level.

  One DLT, lacunar infarction occurred in a cohort A patient. This SAE includes three consecutive SAEs (see above), and muscle weakness that occurs prior to Lacunar infarction is probably a true DLT. Nevertheless, muscle weakness was grade 2, so it does not fit into the previously established definition of DLT. There were no other DLTs in patients treated with eASC, but one severe and possibly treatment-related event (asthenia) was reported in the placebo group, which was sometimes identified by protocol-specified DLTs AEs can lead to misclassification as DLT.

  Lacunar infarction was the only SAE considered to be associated with eASC. Although there was thought to be possible because there was no other obvious cause, the basic mechanism that could have been identified has not been identified. Therefore, a conservative position was taken in the causal assessment.

  As indicated in the literature on MSC, transient fever was the most frequent treatment-related AE that occurred in patients treated with eASC. The mechanism of fever is not clear but may be associated with an acute inflammatory response to MSC in sensitive patients.

  RA patients are at higher risk of serious infections than the general population, and prednisone and biological agents have been shown to increase this risk. The reported risk (RR 95% CI) of serious infections in RA patients treated with anti-TNF therapy ranges from 1.05 (0.9-1.2) to 4.6 (1.8-11.9). In this study, 4 infections were considered related to study treatment and none were severe or severe.

  Thrombotic events have been reported in animal models that received very high doses of iv MSC. However, no venous thrombotic events were reported in this study and no signs suggestive of pulmonary thromboembolism were detected.

  Furthermore, no acute or delayed type hypersensitivity reactions or hematological AEs were reported in patients treated with eASC other than anemia (3 cases).

  So far, clinical experience gained in clinical trials has not shown that the tumorigenicity associated with MSC-based therapy is a major risk. There were no malignant tumors in this study.

  Thus, the results indicate that there are no short-term serious safety issues associated with therapeutic administration of mesenchymal stem cells to RA patients.

Other efficacy endpoints
SF-36 and physical and mental subscales improved in both groups, and there were no statistical differences between the groups. The RA MRI score (RAMRIS) improved from the time of screening to 6 months, but the comparison between treatment groups was not statistically significant.

Example 8
Other DMARD treatments such as cyclosporine, hydroxychloroquine, leflunomide, minocycline, and sulfasalazine, and joints that may have previously received NSAIDs, less than a year after diagnosis and previously treated with at least methotrexate Conduct clinical trials in a rheumatic patient population. When receiving methotrexate, leflunomide, or sulfasalazine, patients must be treated for at least 16 weeks and have been on a continuous basis for at least 4 weeks prior to the start of the study (oral methotrexate ≤ 25 mg / Week; parenteral methotrexate ≦ 20 mg / week; leflunomide ≦ 20 mg / day; sulfasalazine ≦ 3 g / day). When taking oral corticoids, patients must continue to take prednisone ≤ 10 mg / day or equivalent for at least one month prior to screening. If NSAID is being administered, it must be administered continuously for at least 2 weeks prior to screening.

  Furthermore, in order to be eligible for the study, patients must have 4 joints and 4 swellings showing tenderness to palpation based on EULAR DAS28-ESR activity criteria of> 3.2 and 68/66 joints at the time of screening. Must have a joint indicating.

  About half of the patients enrolled in the study receive treatment with a pharmaceutical consisting of a suspension of donor-derived (allogeneic) adipose stromal cells (eASC) in Lactated Ringer's solution. The other half is given a placebo consisting of a suspension of lactated Ringer's solution. Patients in the treatment group are given 3 doses of medication, 4 million cells per kg of patient weight, at 1 week intervals. Patients in the placebo group will receive 3 doses of placebo at 1 week intervals. In either case, administration is by intravenous infusion.

  A pharmaceutical is a cell suspension containing allogeneic adipose-derived stromal cells (eASC) obtained by liposuction from a healthy individual and grown in vitro in a sterile buffer in a disposable vial. Drugs are supplied as sterile, clear, colorless suspensions for intralesional administration provided in 6 mL vials (10 million eASCs per mL of Dulbecco's Modified Eagle Medium [DMEM] containing human serum albumin Suspension). Drugs are suspended in lactated Ringer's solution and then administered at an infusion rate of 4 ml / min.

  The placebo for the study is Lactated Ringer's solution for intralesional administration at an infusion rate of 4 ml / min. A corresponding placebo volume (Lactated Ringer's solution) is administered to each subject in the placebo group. The placebo volume is calculated according to the subject's body weight.

Drug Preparation Allogeneic eASC drugs consist of a cell suspension of living adult stromal cells extracted from the subcutaneous adipose tissue of a healthy donor. Subcutaneous adipose tissue is liposuctioned from a healthy donor and transported to a GMP manufacturing facility. Donations, procurement, and testing are conducted in accordance with the requirements of Directive 2004/23 / EC and therefore under Directives 2006/17 / EC and 2006/83 / EC. ASC is isolated by digesting adipose tissue with type I collagenase followed by centrifugation. The resulting cell pellet is resuspended, lysed with lysed blood, and centrifuged. The stromal vascular fraction resulting from the cell pellet is placed in culture medium and antibiotics in a cell culture vessel and incubated at 37 ° C. and 5% CO 2 and a humid atmosphere. 24-48 hours after seeding, the culture medium is removed to remove the non-adherent cell fraction. ASCs attached to plastic culture plates are increased under in vitro conditions. After reaching 90-95% confluence, the culture medium is changed every 3-4 days, cells are detached with trypsin / EDTA, harvested, centrifuged and increased to the required doubling without adding antibiotics. This is then collected and stored frozen until use. Thaw a sufficient number of cryopreservation vials to prepare the required amount for administration prior to the prescribed administration date. ASC is recovered from the cryopreserved state by seeding and culturing (confirming survival). The culture was washed with phosphate buffer and trypsin / EDTA on the day of filling into vials and packing. ASC is quickly resuspended in selected excipients (Dulbecco's modified Eagle's medium and human albumin serum) to formulate the drug product.

eASC conforms to the same ( Phenotype profile), purity, potency, morphology, viability, and cell growth kinetics.

Claims (21)

  A composition comprising mesenchymal stromal cells for treating rheumatoid arthritis in a subject.   Use of mesenchymal stromal cells in the manufacture of a medicament for treating rheumatoid arthritis in a subject.   A method of treating rheumatoid arthritis in a subject, comprising administering to the subject a composition containing mesenchymal stromal cells.   The disease according to any one of claims 1 to 3, wherein the disease is moderate rheumatoid arthritis with a DAS28 score> 3.2 to ≦ 5.1, a CDAI score> 10 to ≦ 22 and / or a RAPID3 score> 6 to ≦ 12. Composition, use or method.   4. The composition, use or method according to any one of claims 1 to 3, wherein the disease is severe rheumatoid arthritis with a DAS28 score> 5.1, a CDAI score> 22 and / or a RAPID3 score> 12.   6. The composition of any one of claims 1-5, wherein the patient is treated in the acute phase of the disease and / or the patient is first treated within about 6 months from the initial diagnosis or onset of symptoms of the disease, Use or method.   7. A composition, use or method according to any one of claims 1 to 6, wherein MSCs are administered to the subject repeatedly, optionally at weekly intervals.   8. The method of any one of claims 1-7, wherein at least 3 doses of MSC are administered to the subject, e.g., on days 1, 3 and 5 or days 1, 8 and 15 from the initial MSC dose. Composition, use or method.   9. A composition, use or method according to any one of claims 1 to 8, wherein the patient is resistant to treatment with methotrexate.   8. The composition, use or method of claim 7, wherein the patient is resistant to further DMARDs.   9. A composition, use or method according to claim 8, wherein the further DMARD is cyclosporine.   The composition according to any one of claims 1 to 11, wherein the mesenchymal stromal cells are adipose tissue-derived stromal cells, increased mesenchymal stromal cells, or increased adipose tissue-derived stromal cells. , Use or method.   13. A composition, use or method according to any one of claims 1 to 12, wherein the MSC is allogeneic. 14. The composition, use or method according to any one of claims 1 to 13, wherein the composition comprises from about 0.25 x 10 < ⁶ > cells to about 5 x 10 < ⁶ > cells per kg body weight of the subject.   15. At least about 50% of MSCs express one or more of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51, CD54, CD55, CD58, CD59, CD90 and CD105. A composition, use or method according to claim.   At least about 50% of MSCs do not express the markers factor VIII, α-actin, desmin, S-100, keratin, CD11b, CD11c, CD14, CD45, HLAII, CD31, CD34, CD45, STRO-1 and CD133 Item 16. The composition, use or method according to any one of Items 1 to 15.   17. A composition, use or method according to any one of claims 1 to 16, wherein the MSC is administered in a pharmaceutically acceptable carrier and / or diluent.   18. A composition, use or method according to any one of claims 1 to 17, wherein MSC is administered systemically.   2. The MSC is administered via intravenous, intralymphatic, subcutaneous, intradermal, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial routes. A composition, use or method according to any one of -18.   20. A composition, use or method according to any one of claims 1 to 19, wherein the MSC is administered with one or more further therapeutic agents.   21. The composition, use or method of any one of claims 1-20, wherein the MSC is stored frozen.

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